Cell migration is critical for animal development and organogenesis, and inappropriate cell migration contributes to progression of diseases such as metastatic cancer. We are using migration of two specialized C. elegans cells, the distal tip cells (DTC), as an in vivo model system to elucidate the mechanisms by which cells convert extracellular cues to cell migratory behaviors. The genes controlling DTC migration, including metalloproteases, integrins, and Rac GTPases, are strikingly similar to the genes required during metastasis. The goal of the proposed study is to elucidate the mechanism of action of W03H9.4, a novel regulator of in vivo cell migration required for correct timing of DTC turns and correct DTC pathfinding. Our working hypothesis, based on preliminary evidence, is that W03H9.4 controls the cell polarity and directional cell migration of the DTC by regulating the expression pattern or sub-cellular localization of proteins, including netrin guidance cues and/or netrin receptor proteins, which results in altered Rac GTPase signaling and defective cell guidance. First, the cell types and developmental stages that require W03H9.4 for function will be determined through analysis of transgenic animals expressing GFP fusion proteins and immunofluorescence with 1W03H9.4 antibodies. Secondly, the mechanism of W03H9.4 action will be investigated through transgenic rescue of W03H9.4(tm3042) mutant animals, cell-type specific overexpression of W03H9.4, and cell-type specific depletion of W03H9.4 by RNAi. Finally, genetic interactions between W03H9.4, the netrin signaling system, and Rac family GTPases will be investigated, and the effect of disrupted W03H9.4 levels on localization or expression of these signaling proteins will be determined. The exciting connection between W03H9.4 and Rac GTPases gives this study tremendous potential to improve our understanding of the fundamental regulation of cell migration during animal development and in pathologic conditions such as metastatic cancer. PUBLIC HEALTH RELEVANCE: We are using migratory cells in the nematode C. elegans as a model system to study genes involved in cell migration processes, particularly those genes that are relevant to metastatic cancer. This study will determine how a novel gene, W03H9.4, helps cells correctly interpret the extracellular cues that let cells know when to migrate and when to stop migrating. The connection between W03H9.4 and known signaling cascades gives this study tremendous potential to improve our understanding of the fundamental regulation of cell migration during animal development and in pathologic conditions such as metastatic cancer.